Methods for treating cardiac hypertrophy by administering IFN-γ

ABSTRACT

The invention concerns the treatment of cardiac hypertrophy by interferon-gamma (IFN-γ). Cardiac hypertrophy may result from a variety of diverse pathologic conditions, including myocardial infarction, hypertension, hypertrophic cardiomyopathy, and valvular regurgitation. The treatment extends to all stages of the progression of cardiac hypertrophy, with or without structural damage of the heart muscle, regardless of the underlying cardiac disorder.

This is a continuation of application Ser. No. 09/273,099 filed on Mar.19, 1999, now U.S. Pat. No. 6,187,304, which claims priority ofprovisional application Ser. No. 60/080,448 filed on Apr. 2, 1998.

RELATED APPLICATION

This application is a non-provisional application filed under 37 CFR1.53(b)(1), claiming priority under 35 USC 119(e) to provisionalapplication No. 60/080,448 filed 2 Apr. 1998, the contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the effects of IFN-γ oncardiac hypertrophy. More particularly, the invention concerns the useof IFN-γ for the prevention and treatment of cardiac hypertrophy andassociated pathological conditions.

BACKGROUND OF THE INVENTION

Interferon-Gamma (IFN-γ)

Interferons are relatively small, single-chain glycoproteins released bycells invaded by viruses or certain other substances. Interferons arepresently grouped into three major classes, designated leukocyteinterferon (interferon-alpha, α-interferon, IFN-α), fibroblastinterferon (interferon-beta, β-interferon, IFN-β), and immune interferon(interferon-gamma, γ-interferon, IFN-γ). In response to viral infection,lymphocytes synthesize primarily α-interferon (along with a lesseramount of a distinct interferon species, commonly referred to as omegainterferon), while infection of fibroblasts usually inducesβ-interferon. α- and β-interferons share about 20–30 percent amino acidsequence homology. The gene for human IFN-β lacks introns, and encodes aprotein possessing 29% amino acid sequence identity with human IFN-αI,suggesting that IFN-α and IFN-β genes have evolved from a commonancestor (Taniguchi et al., Nature 285, 547–549 [1980]). By contrast,IFN-γ is not induced by viral infection, rather, is synthesized bylymphocytes in response to mitogens, and is scarcely related to theother two types of interferons in amino acid sequence. Interferons-α and-β are known to induce MHC Class I antigens, while IFN-γ induces MHCClass II antigen expression, and also increases the efficiency withwhich target cells present viral peptide in association with MHC Class Imolecules for recognition by cytotoxic T cells.

IFN-γ is a member of the interferon family, which exhibits the antiviraland anti-proliferative properties characteristic of interferons-α and -β(IFN-α and IFN-β) but, in contrast to those interferons, is PH 2 labile.IFN-γ was originally produced upon mitogenic induction of lymphocytes.The recombinant production of human IFN-γ was first reported by Gray,Goeddel and co-workers (Gray et al., Nature 295, 503–508 [1982]), and issubject of U.S. Pat. Nos. 4,762,791, 4,929,544, 4,727,138, 4,925,793,4,855,238, 5,582,824, 5,096,705, 5,574,137, and 5,595,888. Therecombinant human IFN-γ of Gray and Goeddel as produced in E. coli,consisted of 146 amino acids, the N-terminal position of the moleculecommencing with the sequence CysTyrCys. It has later been found that thenative human IFN-γ (i.e., that arising from mitogen induction of humanperipheral blood lymphocytes and subsequent purification) is apolypeptide which lacks the CysTyrCys N-terminus assigned by Gray etal., supra. More recently, the crystal structure of E. coli derivedrecombinant human IFN-γ (rhIFN-γ) was determined (Ealick et al., Science252, 698–702 [1991]), showing that the protein exists as a tightlyintertwined non-covalent homodimer, in which the two identicalpolypeptide chains are oriented in an antiparallel manner.

IFN-γ is known to exhibit a broad range of biological activities,including antitumor, antimicrobial and immunoregulatory activities. Aparticular form of recombinant human IFN-γ (rhIFN-γ-1b, Actimmune®,Genentech, Inc. South San Francisco, Calif.) is commercially availableas an immunomodulatory drug for the treatment of chronic granulomatousdisease characterized by severe, recurrent infections of the skin, lymphnodes, liver, lungs, and bones due to phagocyte disfunction (Baehner, R.L., Pediatric Pathol. 10, 143–153 [1990]). IFN-γ has also been proposedfor the treatment of atopic dermatitis, a common inflammatory skindisease characterized by severe pruritus, a chronically relapsing coursewith frequent periods of exacerbation, a distinctive clinical morphologyand distribution of skin lesions (see PCT Publication No. WO 91/07984published 13 Jun. 1991), vascular stenosis, including the treatment ofrestenosis following angioplasty and/or vascular surgery (PCTPublication No. WO 90/03189 published 5 Apr. 1990), various lungconditions, including respiratory distress syndromes (RDS), such asadult respiratory distress syndrome (ARDS) and a neonatal form, termedvariously as idiopathic RDS or hyaline membrane disease (PCT PublicationNo. WO 89/01341, published 23 Feb. 1989). In addition, IFN-γ has beenproposed for use in the treatment of various allergies, e.g. asthma, andHIV-infection-related conditions, such as opportunistic infections, e.g.Pneumocystis carinii pneumonia, and trauma-associated sepsis. ImpairedIFN-γ production has been observed in multiple-sclerosis (MP) patients,and it has been reported that the production of IFN-γ is greatlysuppressed in suspensions of mitogen-stimulated mononuclear cellsderived from AIDS patients. For a review see, for example, Chapter 16,“The Presence of Possible Pathogenic Role of Interferons in Disease”,In: Interferons and other Regulatory Cytokines, Edward de Maeyer (1988,John Wilet and Sons Publishers).

Interferon-γ, along with other cytokines, has been implicated as aninducer of inducible nitric oxide (iNOS) which, in turn, has beendescribed as an important mediator of the inflammatory mechanismunderlying heart failure, of the cardiac response to sepsis or allograftrejection, as well as the progression of dilated cardiomyopathies ofdiverse etiologies. Ungureanu-Longrois et al., Circ. Res. 77, 494–502(1995); Pinsky et al., J. Clin. Invest. 95, 677–685 (1995); Singh etal., J. Biol. Chem. 270, 28471–8 (1995); Birks and Yacoub, CoronaryArtery Disease 8, 389–402 (1997); Hattori et al., J. Mol. Cell. Cardiol.29, 1585–92 (1,997). Indeed, IFN-γ has been reported to be the mostpotent single cytokine with regard to myocyte iNOS induction (Watkins etal., J. Mol. & Cell. Cardiol. 27, 2015–29 [1995]).

Cardiac Hypertrophy

Hypertrophy is generally defined as an increase in size of an organ orstructure independent of natural growth that does not involve tumorformation. Hypertrophy of an organ or tissue is due either to anincrease in the mass of the individual cells (true hypertrophy), or toan increase in the number of cells making up the tissue (hyperplasia),or both.

Cardiac hypertrophy is the enlargement of heart that is activated byboth mechanical and hormonal stimuli and enables the heart to adapt todemands for increased cardiac output or to injury. Morgan and Baker,Circulation 83, 13–25 (1991). This response is frequently associatedwith a variety of distinct pathological conditions, such ashypertension, aortic stenosis, myocardial infarction, cardiomyopathy,valvular regurgitation, cardiac shunt, congestive heart failure, etc.

On a cellular level, the heart functions as a syncytium of myocytes andsurrounding support cells, called non-myocytes. While non-myocytes areprimarily fibroblast/mesenchymal cells, they also include endothelialand smooth muscle cells. Indeed, although myocytes make up most of theadult myocardial mass, they represent only about 30% of the total cellnumbers present in heart.

The enlargement of embryonic heart is largely dependent on an increasein myocyte number, which continues until shortly after birth, whencardiac myocytes lose their proliferative capacity. Further growthoccurs through hypertrophy of the individual cells. Hypertrophy of adultcardiac ventricular myocytes is a response to a variety of conditionswhich lead to chronic hemodynamic overload. Thus, in response tohormonal, physiological, hemodynamic, and pathological stimuli, adultventricular muscle cells can adapt to increased workloads through theactivation of a hypertrophic process. This response is characterized byan increase in myocyte cell size and contractile protein content ofindividual cardiac muscle cells, without concomitant cell division andactivation of embryonic genes, including the gene for atrial natriureticpeptide (ANP). Chien et al., FASEB J. 5, 3037–3046(1991); Chien et al.,Annu. Rev. Physiol. 55, 77–95 (1993). An increment in myocardial mass asa result of an increase in myocyte size that is associated with anaccumulation of interstitial collagen within the extracellular matrixand around intramyocardial coronary arteries has been described in leftventricular hypertrophy secondary to pressure overload in humans(Caspari et al., Cardiovasc. Res. 11, 554–8 [1977]; Schwarz et al., Am.J. Cardiol. 42, 895–903 [1978]; Hess et al., Circulation 63, 360–371[1981]; Pearlman et al., Lab. Invest. 46, 158–164 [1982]). Cardiachypertrophy due to chronic hemodynamic overload is the common end resultof most cardiac disorders and a consistent feature of cardiac failure.

It has also been suggested that paracrine factors produced bynon-myocyte supporting cells may additionally be involved in thedevelopment of cardiac hypertrophy, and various non-myocyte derivedhypertrophic factors, such as, leukocyte inhibitory factor (LIF) andendothelin, have been identified. Metcalf, Growth Factors 7, 169–173(1992); Kurzrock et al., Endocrine Reviews 12, 208–217 (1991); Inoue etal., Proc. Natl. Acad. Sci. USA 86: 2863–2867 (1989); Yanagisawa andMasaki, Trends Pharm. Sci. 10, 374–378 (1989); U.S. Pat. No. 5,573,762(issued Nov. 12, 1996). Further exemplary factors that have beenidentified as potential mediators of cardiac hypertrophy includecardiotrophin-1 (CT-1) (Pennica et al., Proc. Nat. Acad. Sci. USA92:1142–1146 [1995]), catecholamines, adrenocorticosteroids,angiotensin, and prostaglandins.

Adult myocyte hypertrophy is initially beneficial as a short termresponse to impaired cardiac function by permitting a decrease in theload on individual muscle fibers. With severe, long-standing overload,however, the hypertrophied cells begin to deteriorate and die. Katz,“Heart Failure”, in: Katz A. M. ed., Physiology of the Heart (New York,Raven Press, 1992) pp. 638–668. Cardiac hypertrophy is a significantrisk factor for both mortality and morbidity in the clinical course ofheart failure. Katz, Trends Cardiovasc. Med. 5, 37–44 (1995).

For further details of the causes and pathology of cardiac hypertrophysee, e.g. Heart Disease, A Textbook of Cardiovascular Medicine,Braunwald, E. ed., W.B. Saunders Co., 1988, Chapter 14, Pathophysiologyof Heart Failure.

Treatment of Cardiac Hypertrophy

At present, the treatment of cardiac hypertrophy varies depending on theunderlying cardiac disease. Catecholamines, adrenocorticosteroids,angiotensin, prostaglandins, leukemia inhibitory factor (LIF),endothelin (including endothelin-1, -2, and -3 and big endothelin),cardiotrophin-1 (CT-1) and cardiac hypertrophy factor (CHF) are amongthe factors identified as potential mediators of hypertrophy.

For example, β-adrenergic receptor blocking drugs (β-blockers, e.g.,propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol,penbutolol, acetobutolol, atenolol, metoprolol, carvedilol, etc.) andverapamil have been used extensively in the treatment of hypertrophiccardiomyopathy. The beneficial effects of β-blockers on symptoms (e.g.chest pain) and exercise tolerance are largely due to a decrease in theheart rate with a consequent prolongation of diastole and increasedpassive ventricular filling. Thompson et al., Br. Heart J. 44, 488–98(1980); Harrison et al., Circulation 29, 84–98 (1964). Verapamil hasbeen described to improve ventricular filling and probably reducingmyocardial ischemia. Bonow et al., Circulation 72, 853–64 (1985).Nifedipine and diltiazem have also been used occasionally in thetreatment of hypertrophic cardiomyopathy. Lorell et al., Circulation 65,499–507 (1982); Betocchi et al., Am. J. Cardiol. 78, 451–7 (1996).However, because of its potent vasodilating properties, nifedipine maybe harmful, especially in patients with outflow obstruction.Disopyramide has been used to relieve symptoms by virtue of its negativeinotropic properties. Pollick, N. Engl. J. Med. 307, 997–9 (1982). Inmany patients, however, the initial benefits decrease with time. Wigleet al., Circulation 92, 1680–92 (1995).

Antihypertensive drug therapy has been reported to have beneficialeffects on cardiac hypertrophy associated with elevated blood pressure.Examples of drugs used in antihypertensive therapy, alone or incombination, are calcium antagonists, e.g. nitrendipine; β-adrenergicreceptor blocking agents, e.g., those listed above; angiotensinconverting enzyme (ACE) inhibitors, e.g., quinapril, captopril,enalapril, ramipril, benazepril, fosinopril, lisinopril; diuretics, e.g.chorothiazide, hydrochlorothiazide, hydroflumethazide,methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide,indapamide; calcium channel blockers, e.g. diltiazem, nifedipine,verapamil, nicardipine. For example, treatment of hypertension withdiltiazem and captopril showed a decrease in left ventricular musclemass, but the Doppler indices of diastolic function did not normalize.Szlachcic et al., Am. J. Cardiol. 63, 198–201 (1989); Shahi et al.,Lancet 336, 458–61 (1990). These findings were interpreted to indicatethat excessive amounts of interstitial collagen may remain afterregression of left ventricular hypertrophy. Rossi et al., Am. Heart J.124, 700–709 (1992). Rossi et al., supra, investigated the effect ofcaptopril on the prevention and regression of myocardial cellhypertrophy and interstitial fibrosis in pressure overload cardiachypertrophy, in experimental rats.

As there is no generally applicable therapy for the treatment of cardiachypertrophy, the identification of factors that can prevent or reducecardiac myocyte hypertrophy is of primary importance in the developmentof new therapeutic strategies to inhibit pathophysiological cardiacgrowth.

SUMMARY OF THE INVENTION

We have unexpectedly found that IFN-γ inhibits the prostaglandin F_(2α)(PGF_(2α))- and phenylephrine-induced spreading of cardiac myocytesisolated from adult rats. We have further found that IFN-γ inhibits invivo both cardiac hypertrophy induced by fluprostenol, an agonist analogof PGF_(2α), and hypertrophy induced by pressure overload in a ratmodel.

Accordingly, the present invention concerns the treatment of cardiachypertrophy, regardless of the underlying cause, by administering atherapeutically effective dose of IFN-γ. If the objective is thetreatment of human patients, IFN-γ preferably is recombinant human IFN-γ(rhIFN-γ), most preferably, rhIFN-γ-1b, which will be definedhereinbelow. The concept of treatment is used in the broadest sense, andspecifically includes the prevention (prophylaxis), moderation,reduction, and curing of cardiac hypertrophy of any stage.

IFN-γ preferably is administered in the form of a liquid pharmaceuticalformulation, which may be preserved to achieve extended storagestability. Preserved liquid pharmaceutical formulations might containmultiple doses of IFN-γ, and might, therefore, be suitable for repeateduse.

IFN-γ might be administered in combination with one or more furthertherapeutic agent used for the treatment of cardiac hypertrophy, or aphysiological condition instrumental in the development of cardiachypertrophy, such as elevated blood pressure, aortic stenosis, ormyocardial infarction.

The invention further concerns a method for making a pharmaceuticalcomposition for the treatment of cardiac hypertrophy, which comprisesIFN-γ as an active ingredient.

The invention also concerns a pharmaceutical product which comprises:

-   -   (a) a pharmaceutical composition comprising at least one        therapeutically effective dosage of IFN-γ;    -   (b) a container containing said pharmaceutical composition; and    -   (c) a label affixed to said container, or a package insert        included in said pharmaceutical product referring to the use of        said IFN-γ in the treatment of cardiac hypertrophy.

BRIEF DESCRIPTION OF THE FIGURES

In the Figures and throughout the examples, “IFN” or “IFN-γ” refers torecombinant mouse IFN-γ (Genentech, Inc., South San Francisco, Calif.,or Genzyme, Cambridge, Mass.).

FIGS. 1A–1F Inhibition of prostaglandin F_(2α) (PGF_(2α))-inducedspreading response by IFN-γ. Myocytes were pre-incubated with salinevehicle or IFN-γ (500 U/ml) on day of isolation. A second addition ofvehicle or IFN-γ was performed 24h after isolation, along with theaddition of either vehicle or PGF_(2α) (10⁻⁷ M). After an additional 72hr incubation, cells were fixed in glutaraldehyde, stained with eosin Yand viewed by fluorescence microscopy. A, B, C Cardiac myocytes after 4days in culture: control, PGF_(2α), and PGF_(2α)+IFN-γ, respectively. D,E, F Histographs showing maximum breadth of rod shaped cardiac myocytesversus percent frequency of breadth occurrence. The maximum breadth ofrod-shaped cells was determined by fluorescence microscopy and imagingsoftware. At least 200 rod shaped cells from a single experiment wereexamined per group. IFN-γ alone had no observable effect on themorphology of the cells. P<0.001 for all group comparisons.

FIGS. 2A–2E Dose responsive inhibition of PGF_(2α)-induced response byIFN-γ (500–25 U/ml). Myocytes were pre-incubated with saline vehicle orIFN-γ on day of isolation. A second amount of IFN-γ was added 24 hrafter isolation, along with the addition of either vehicle or PGF_(2α)(10⁻⁷ M). After an additional 72 hr incubation, cells were fixed inglutaraldehyde, stained with eosin Y and viewed under fluorescence.Quantitation of myocyte morphology: A control, B PGF_(2α), CPGF_(2α)+IFN-γ (25 U/ml), D PGF_(2α)+IFN-γ (100 U/ml), E PGF_(2α)+IFN-γ(500 U/ml). Histographs showing maximum breadth of rod shaped cardiacmyocytes versus percent frequency of breadth occurrence. The maximumbreadth of rod-shaped cells was determined by fluorescence microscopyand imaging software. At least 200 rod shaped cells from a singleexperiment were examined per group. IFN-γ alone had no observable effecton the morphology of the cells. P<0.001 for all group comparisons.

FIGS. 3A–3F Inhibition of phenylephedrine (PE)-induced spreadingresponse by IFN-γ. Myocytes were pre-incubated with saline vehicle orIFN-γ (500 U/ml) on day of isolation. A second addition of vehicle orIFN-γ was performed 24h after isolation, along with the addition ofeither vehicle or PE (10⁻⁵ M). After an additional 72 hr incubation,cells were fixed in glutaraldehyde, stained with eosin Y and viewed byfluorescence microscopy. A, B, C Cardiac myocytes after 4 days inculture: control, PE, and PE+IFN-γ, respectively. D, E, F Histographsshowing maximum breadth of rod shaped cardiac myocytes versus percentfrequency of breadth occurrence. The maximum breadth of rod-shaped cellswas determined by fluorescence microscopy and imaging software. At least200 rod shaped cells from a single experiment were examined per group.IFN-γ alone had no observable effect on the morphology of the cells.P<0.001 for all group comparisons.

FIGS. 4A–4C Effects of IFN-γ on cardiac hypertrophy induced byfluprostenol in rats. Data are presented as mean±SEM. The number inparenthesis is the number of animals in each group. *P<0.05, *P<0.01,compared to the vehicle group. #P<0.05, ##P<0.01, compared to the Flupgroup. Flup: fluprostenol; IFN=IFN-γ; HW: heart weight; BW: body weight;VW: ventricular weight; LVW: left ventricular weight.

FIGS. 5A–5B Effects of Flup and/or IFN on MAP and HR. Data are presentedas mean±SEM. The number in parenthesis is the number of animals in eachgroup. *P<0.05, compared to the vehicle group. #P<0.05, compared to theFlup group. +P<0.05, compared to the Flup+IFN group. Flup: fluprostenol;IFN: IFN-γ; MAP: mean arterial pressure; HR: heart rate.

FIGS. 6A–6D Bargraphs showing the effect of fluprostenol (FLUP) andIFN-γ on: A Skeletal actin (SKA); B Sarcoplasmic reticulum calciumATPase (SRCA); C Collagen I (COL I); D Atrial natriuretic factor (ANF)expression. Expression levels are normalized toglyceraldehyde-3-phosphate dehydrogenase (GAPDH) message. VEH isvehicle. There were 7 animals per group and the data are presented asthe mean±SEM. P<0.05 vs VEH group.

FIGS. 7A–7C Effects of IFN-γ on heart weight, ventricular weight, andleft ventricular weight in rats with pressure overload. Data arepresented as mean±SEM. The number in parenthesis is the number ofanimals in each group. **P<0.01, compared to the sham group. ##P<0.01,compared to the Banded+vehicle group. Sham: sham-operated rats; Banded:aortic-banded rats; IFN: IFN-γ; HW: heart weight; VW: ventricularweight: LVW: left ventricular weight.

FIGS. 8A–8C Effects of IFN-γ on the ratio of heart weight, ventricularweight, and left ventricular weight to body weight in rats with pressureoverload. Data are presented as mean±SEM. The number in parenthesis isthe number of animals in each group. **P<0.01, compared to the shamgroup. ##P<0.01, compared to the Banded+vehicle group. Sham:sham-operated rats; Banded: aortic-banded rats: IFN: IFN-γ; HW: heartweight: BW: body weight; VW: ventricular weight; LVW: left ventricularweight.

FIGS. 9A–9C Effects of IFN-γ on systolic arterial pressure, meanarterial pressure, and diastolic arterial pressure in rats with pressureoverload. The number in parenthesis is the number of animals in eachgroup. **P<0.01, compared to the sham group. Sham: sham-operated rats;Banded: aortic-banded rats; IFN: IFN-γ, SAP: systolic arterial pressure;MAP: mean arterial pressure; DAP: diastolic arterial pressure.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

“Gamma interferon”, “interferon-gamma”, or “IFN-γ” refers variously toall forms of (human and non-human animal) gamma interferon that areshown to be biologically active in any assay of cardiac hypertrophy,e.g. the hypertrophy assays disclosed herein, and is meant to include,but is not limited to, mature, pro, met and/or des(1–3) (also referredto as desCysTyrCys IFN-γ) form, whether obtained from natural source,chemically synthesized or produced by techniques of recombinant DNAtechnology. A complete description of the preparation of recombinanthuman IFN-γ (rhuIFN-γ) including its cDNA and amino acid sequences aredisclosed, for example, in U.S. Pat. Nos. 4,727,138; 4,762,791;4,925,793; 4,929,554; 5,582,824; 5,096,705; 4,855,238; 5,574,137; and5,595,888. CysTyrCys-lacking recombinant human IFN-γ, includingvariously truncated derivatives are, for example, disclosed in EuropeanPatent Publication No. 146,354. Non-human animal interferons, includingIFN-γ, are, for example, disclosed in European Publication No. 88,622.The term includes variously glycosylated forms and other variants (e.g.amino acid sequence variants) and derivatives of such native (wild-type)interferons, whether known in the art or will become available in thefuture. Examples of such variants are alleles, and the products ofsite-directed mutagenesis in which residues are deleted, inserted and/orsubstituted (see, e.g. European Publication No. 146,354 referred toabove). IFN-γ is known to have a narrow host range, therefore, IFN-γhomologous to the animal to be treated should be used. In human therapy,the desCysTyrCys variant of the sequence shown in U.S. Pat. No.4,717,138 and its counterpart EP 77,670, is preferably employed, andoptionally the C-terminal variant in which the last four amino acidresidues are deleted in post-translational processing. For humantherapeutic use, the IFN-γ of the present invention preferably isrecombinant human IFN-γ (rhIFN-γ), with or without the amino acidsCysTyrCys at the N-terminus. More preferably, IFN-γ is a recombinanthuman IFN-γ species (recombinant human interferon gamma-1b, rhIFN-γ-1b,containing 140 amino acids), which is the active ingredient of thecommercial formulation, Actimmune® (Genentech, Inc., South SanFrancisco, Calif.). As IFN-γ is known to be highly species specific, inanimal experiments, or for veterinary use. IFN-γ of the animal speciesto be treated is preferably employed. Thus, in the in vivo experimentsusing a rat animal model, murine (mouse) recombinant IFN-γ (Genentech,Inc.) has been employed. Rat and mice and sufficiently closely relatedto permit the use of mouse IFN-γ in a rat model.

In a pharmacological sense, in the context of the present invention, a“therapeutically effective amount” of IFN-γ refers to an amounteffective in the treatment of hypertrophy, specifically cardiachypertrophy.

“Hypertrophy”, as used herein, is defined as an increase in mass of anorgan or structure independent of natural growth that does not involvetumor formation. Hypertrophy of an organ or tissue is due either to anincrease in the mass of the individual cells (true hypertrophy), or toan increase in the number of cells making up the tissue (hyperplasia),or both. Certain organs, such as the heart, lose the ability to divideshortly after birth. Accordingly, “cardiac hypertrophy” is defined as anincrease in mass of the heart, which, in adults, is characterized by anincrease in myocyte cell size and contractile protein content withoutconcomitant cell division. The character of the stress responsible forinciting the hypertrophy, (e.g., increased preload, increased afterload,loss of myocytes, as in myocardial infarction, or primary depression ofcontractility), appears to play a critical role in determining thenature of the response. The early stage of cardiac hypertrophy isusually characterized morphologically by increases in the size ofmycrofibrils and mitochondria, as well as enlargement of mitochondriaand nuclei. At this stage, while muscle cells are larger than normal,cellular organization is largely preserved. At a more advanced stage ofcardiac hypertrophy, there are preferential increases in the size ornumber of specific organelles, such a mitochondria, and new contractileelements are added in localized areas of the cells, in an irregularmanner. Cells subjected to long-standing hypertrophy show more obviousdisruptions in cellular organization, including markedly enlarged nucleiwith highly lobulated membranes, which displace adjacent myofibrils andcause breakdown of normal Z-band registration. The phrase “cardiachypertrophy” is used to include all stages of the progression of thiscondition, characterized by various degrees of structural damage of theheart muscle, regardless of the underlying cardiac disorder.

“Heart failure” refers to an abnormality of cardiac function where theheart does not pump blood at the rate needed for the requirements ofmetabolizing tissues. The heart failure can be caused by a number offactors, including ischemic, congenital, rheumatic, or idiopathic forms.

“Congestive heart failure” is a progressive pathologic state where theheart is increasingly unable to supply adequate cardiac output (thevolume of blood pumped by the heart over time) to deliver the oxygenatedblood to peripheral tissues. As congestive heart failure progresses,structural and hemodynamic damages occur. While these damages have avariety of manifestations, one characteristic symptom is ventricularhypertrophy. Congestive heart failure is a common end result of a numberof various cardiac disorders.

“Myocardial infarction” generally results from atherosclerosis of thecoronary arteries, often with superimposed coronary thrombosis. It maybe divided into two major types: transmural infarcts, in whichmyocardial necrosis involves the full thickness of the ventricular wall,and subendocardial (nontransmural) infarcts, in which the necrosisinvolves the subendocardium, the intramural myocardium, or both, withoutextending all the way through the ventricular wall to the epicardium.Myocardial infarction is known to cause both a change in hemodynamiceffects and an alteration in structure in the damaged and healthy zonesof the heart. Thus, for example, myocardial infarction reduces themaximum cardiac output and the stroke volume of the heart. Alsoassociated with myocardial infarction is a stimulation of the DNAsynthesis occurring in the interstice as well as an increase in theformation of collagen in the areas of the heart not affected.

As a result of the increased stress or strain placed on the heart inprolonged hypertension due, for example, to the increased totalperipheral resistance, cardiac hypertrophy has long been associated with“hypertension”. A characteristic of the ventricle that becomeshypertrophic as a result of chronic pressure overload is an impaireddiastolic performance. Fouad et al., J. Am. Coll. Cardiol. 4, 1500–6(1984); Smith et al., J. Am. Coll. Cardiol. 5, 869–74 (1985). Aprolonged left ventricular relaxation has been detected in earlyessential hypertension, in spite of normal or supranormal systolicfunction. Hartford et al., Hypertension 6, 329–338 (1984). However,there is no close parallelism between blood pressure levels and cardiachypertrophy. Although improvement in left ventricular function inresponse to antihypertensive therapy has been reported in humans,patients variously treated with a diuretic (hydrochlorothiazide), aβ-blocker (propranolol), or a calcium channel blocker (diltiazem), haveshown reversal of left ventricular mass, without improvement indiastolic function. Inouye et al., Am. J. Cardiol. 53, 1583–7 (1984).

Another complex cardiac disease associated with cardiac hypertrophy is“hypertrophic cardiomyopathy”. This condition is characterized by agreat diversity of morphologic, functional, and clinical features (Maronet al., N. Engl. J. Med. 316, 780–9 [1987]; Spirito et al., N. Engl. J.Med. 320, 749–55 [1989]; Louie and Edwards, Prog. Cardiovasc. Dis. 36,275–308 [1994]; Wigle et al., Circulation 92, 1680–92 [1995]), theheterogeneity of which is accentuated by the fact that it afflictspatients of all ages (Spirito et al., N. Engl. J. Med. 336, 775–785[1997]). The causative factors of hypertrophic cardiomyopathy are alsodiverse and little understood. Recent data suggest that β-myosin heavychain mutations may account for approximately 30 to 40 percent of casesof familial hypertrophic cardiomyopathy (Watkins et al., N. Engl. J.Med. 326, 1108–14 [1992] Schwartz et al., Circulation 91, 532–40 [1995];Marian and Roberts. Circulation 92, 1336–47 [1995]; Thierfelder et al.,Cell 77, 701–12 [1994]; Watkins et al., Nat. Gen. 11, 434–7 [1995]).

Supravalvular “aortic stenosis” is an inherited vascular disorder, thatis characterized by narrowing of the ascending aorta, but otherarteries, including the pulmonary arteries, may also be affected.Untreated aortic stenosis may lead to increased intracardiac pressureresulting in myocardial hypertrophy and eventually heart failure anddeath. The pathogenesis of this disorder is not fully understood, buthypertrophy and possibly hyperplasia of medial smooth muscle areprominent features of this disorder. It has been reported that molecularvariants of the elastin gene are involved in the development andpathogenesis of aortic stenosis. (U.S. Pat. No. 5,650,282 issued Jul.22, 1997.)

“Valvular regurgitation” occurs as a result of heart diseases resultingin disorders of the cardiac valves. Various diseases, like rheumaticfever, can cause the shrinking or pulling apart of the valve orifice,while other diseases may result in endocarditis, an inflammation of theendocardium or lining membrane of the atrioventricular orifices andoperation of the heart. Defects such as the narrowing of the valvestenosis or the defective closing of the valve result in an accumulationof blood in the heart cavity or regurgitation of blood past the valve.If uncorrected, prolonged valvular stenosis or insufficiency may resultin cardiac hypertrophy and associated damage to the heart muscle, whichmay eventually necessitate valve replacement.

The treatment of all these, and other cardiac disorders accompanied bycardiac hypertrophy is subject of the present invention.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) hypertrophy. Those in need of treatment include those alreadywith the disorder as well as those prone to have the disorder or thosein whom the disorder is to be prevented. The hypertrophy may result fromany cause, including idiopathic, cardiotrophic, or myotrophic causes, orischemia or ischemic insults, such as myocardial infarction.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial antihypertrophic effect for an extended period of time.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, cats, cows, horses, sheep, pigs, etc.Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

B. Modes of Carrying out the Invention

1. Cardiac Hypertrophy Assays

In Vitro Assays

a. Induction of Spreading of Adult Rat Cardiac Myocytes

In this assay, ventricular myocytes are isolated from a single (maleSprague-Dawley) rat, essentially following a modification of theprocedure described in detail by Piper et al., “Adult ventricular ratheart muscle cells.” In: Cell Culture Techniques in Heart and VesselResearch, H. M. Piper, ed., Berlin: Spinger-Verlag, 1990, pp. 36–60.This procedure permits the isolation of adult ventricular myocytes andthe long-term culture of these cells in the rod-shaped phenotype.Phenylephrine and Prostaglandin F_(2α), (PGF_(2α)) have been shown toinduce a spreading response in these adult cells. Piper et al., supra;Lai et al., Am J. Physiol. 1996; 271 (Heart Circ. Phvsiol.40):H2197–H2208. The inhibition of myocyte spreading induced byPGF_(2α), or PGF_(2α) analogs, (e.g. fluprostenol) and phenylephrine byvarious potential inhibitors of cardiac hypertrophy is then tested. Adetailed protocol is described in the examples that follow.

In Vivo Assays

a. Inhibition of Cardiac Hypertrophy Induced by Fluprostenol In Vivo

This pharmacological model tests the ability of IFN-γ to inhibit cardiachypertrophy induced in rats (e.g. male Wistar or Sprague-Dawley) bysubcutaneous injection of fluprostenol (an agonist analog of PGF_(2α)).It is known that rats with pathologic cardiac hypertrophy induced bymyocardial infarction have chronically elevated levels of extractablePGF_(2α) in their myocardium. Lai et al, Am. J. Physiol. (Heart Circ.Physiol.) 271:H2197–H2208 (1996). Accordingly, factors that can inhibitthe effects of fluprostenol on myocardial growth in vivo are potentiallyuseful for treating cardiac hypertrophy. The effects of IFN-γ on cardiachypertrophy are determined by measuring the weight of heart, ventricles,and left ventricle (normalized by body weight) relative tofluprostenol-treated rats not receiving IFN-γ. A detailed description ofthis assay is provided in the examples.

b. Pressure-Overload Cardiac Hypertrophy Assay.

For in vivo testing it is common to induce pressure-overload cardiachypertrophy by constriction of the abdominal aorta of test animals. In atypical protocol rats (e.g. male Wistar or Sprague-Dawley) are treatedunder anesthesia, and the abdominal aorta of each rat is narrowed downjust below the diaphragm. Beznak M., Can. J. Biochem. Physiol. 33,985–94 (1955). The aorta is exposed through a surgical incision, and ablunted needle is placed next to the vessel. The aorta is constrictedwith a ligature of wool thread around the needle, which is immediatelyremoved and which reduces the lumen of the aorta to the diameter of theneedle. This approach is described, for example, in Rossi et al., Am.Heart J. 124, 700–709 (1992) and O'Rourke and Reibel, P.S.E.M.B. 200,95–100 (1992). A detailed description of the protocol used by thepresent inventors is disclosed in the examples hereinbelow.

b. Effect on Cardiac Hypertrophy Following Experimentally InducedMyocardial Infarction (MI).

Acute MI is induced in rats by left coronary artery ligation andconfirmed by electrocardiographic examination. A sham-operated group ofanimals is also prepared as control animals. Earlier data have shownthat cardiac hypertrophy is present in the group of animals with MI, asevidenced by an 18% increase in heart weight-to-body weight ratio. Laiet al., supra. Treatment of these animals with candidate blockers ofcardiac hypertrophy, e.g. IFN-γ provides valuable information about thetherapeutic potential of the candidates tested.

2. Uses, Therapeutic Compositions and Administration of IFN-γ

In accordance with the present invention, IFN-γ can be used for thetreatment of cardiac hypertrophy, i.e. the enlargement of heart,regardless of the etiology and pathogenesis. When an excessive pressureor volume load is imposed on the heart (ventricle), cardiac (myocardial)hypertrophy develops, providing a fundamental compensatory mechanismthat permits the ventricle to sustain its burden. Krayenbuehl et al.,Eur. Heart J. 4 (Suppl. A), 29 (1983). The character of the stress(increased preload, increased afterload, loss of myocytes, as inmyocardial infarction, or primary depression of contractility)responsible of the development of hypertrophy plays a critical role indetermining the nature of the hypertrophic response. Scheuer andButtrick, Circulation 75(Suppl. I), 63 (1987). The present inventionconcerns the treatment of cardiac hypertrophy associated with anyunderlying pathological condition, including, without limitation, postmyocardial infarction, hypertension; aortic stenosis, cardiomyopathy,valvular regurgitation, cardiac shunt, and congestive heart failure. Themain characteristics of these conditions have been discussedhereinabove.

Particularly important is the use of IFN-γ for the prevention of cardiacfailure following myocardial infarction. About 750,000 patients sufferfrom acute myocardial infarction (AMI) annually, and approximatelyone-fourth of all death in the United States are due to AMI. In recentyears, thrombolytic agents, e.g. streptokinase, urokinase, and inparticular tissue plasminogen activator (t-PA) have significantlyincreased the survival of patients who suffered myocardial infarction.When administered as a continuous intravenous infusion over 1.5 to 4hours, t-PA produces coronary patency at 90 minutes in 69% to 90% of thetreated patients. Topol et al., Am. J. Cardiol. 61, 723–8 (1988);Neuhaus et al., J. Am. Coll. Cardiol. 12, 581–7 (1988); Neuhaus et al.,J. Am. Col. Cardiol. 14, 1566–9 (1989). The highest patency rates havebeen reported with high dose or accelerated dosing regimens. Topol, J.Am. Coll. Cardiol. 15, 922–4 (1990). t-PA may also be administered as asingle bolus, although due to its relatively short half-life, it isbetter suited for infusion therapy. Tebbe et al., Am. J. Cardiol. 64448–53 (1989). A t-PA variant, specifically designed to havelongerhalf-life and very high fibrin specificity, TNK t-PA (a T103N,N117Q, KHRR(296–299)AAAA t-PA variant, Keyt et al., Proc. Natl. Acad.Sci. USA 91, 3670–3674 (1994)) is particularly suitable for bolusadministration. However, despite all these advances, the long-termprognosis of patient survival depends greatly on the post-infarctionmonitoring and treatment of the patients, which should includemonitoring and treatment of cardiac hypertrophy.

Another important therapeutic indication is the treatment of cardiachypertrophy associated with hypertension. As noted before, sustainedhypertension is known to result in cardiac hypertrophy. Although certainhypotensive agents have been shown to reduce left ventricular mass,treatment does not always resulted in the improvement of diastolicfunction. Accordingly, IFN-γ can be administered in combination withβ-adrenergic receptor blocking agents, e.g., propranolol, timolol,tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol,atenolol, metoprolol, carvedilol: angiotensin converting enzyme (ACE)inhibitors, e.g., quinapril, captopril, enalapril, ramipril, benazepril,fosinopril, lisinopril; diuretics, e.g. chorothiazide,hydrochlorothiazide, hydroflumethazide, methylchlothiazide,benzthiazide, dichlorphenamide, acetazolamide, indapamide; and/orcalcium channel blockers, e.g., diltiazem, nifedipine, verapamil,nicardipine. Pharmaceutical compositions comprising the therapeuticagents identified here by their generic names are commerciallyavailable, and are to be administered following the manufacturers'instructions for dosage, administration, adverse effects,contraindications, etc. (See, e.g. Physicians' Desk Reference, MedicalEconomics Data Production Co. Montvale, N.J., 51th Edition, 1997.)

IFN-γ may also be administered prophylactically to patients with cardiachypertrophy, to prevent the progression of the condition, and avoidsudden death, including death of asymptomatic patients. Suchpreventative therapy is particularly warranted in the case of patientsdiagnosed with massive left ventricular cardiac hypertrophy (a maximalwall thickness of 35 mm or more in adults, or a comparable value inchildren), or in instances when the hemodynamic burden on the heart isparticularly strong.

IFN-γ may also be useful in the management of atrial fibrillation, whichdevelops in a substantial portion of patients diagnosed withhypertrophic cardiomyopathy.

IFN-γ is administered in the form of a pharmaceutical compositioncomprising IFN-γ as an active ingredient, in conjunction with apharmaceutically acceptable carrier. Therapeutic formulations of IFN-γfor treating cardiac hypertrophy are prepared for storage by mixingIFN-γ having the desired degree of purity with optional physiologicallyacceptable carriers, excipients, or stabilizers (Remington'sPharmaceutical Sciences, supra), in the form of lyophilized cake oraqueous solutions. Acceptable carriers, excipients, or stabilizers arenon-toxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics, or polyethylene glycol (PEG).

IFN-γ to be used for in vivo administration must be sterile. This isreadily accomplished by filtration through sterile filtration membranes,prior to or following lyophilization and reconstitution. IFN-γordinarily will be stored in lyophilized form or in solution.

IFN-γ may be used in lyophilized form, in combination with otheringredients for reconstitution with an appropriate diluent at the timefor use. Because IFN-γ is known to be acid labile, it has traditionallybeen handled at neutral or slightly alkaline pH. See, for example, U.S.Pat. No. 4,499,014 which discloses reactivation of a lyophilized acidicIFN-γ solution to a pH of 6 to 9. Neutral or slightly alkaline solutionsof higher concentrations of IFN-γ are generally unsuitable as injectableformulations because of the immediate formation of a visibleprecipitate. Such precipitate may cause thrombosis on administration ordecrease potency. European Patent Publication No. 0196,203 disclosesreconstitution of lyophilized IFN-γ to a pH of 4 to 6.0.

Stable liquid pharmaceutical compositions comprising an effective amountof non-lyophilized IFN-γ along with a buffer capable of maintaining thepH at 4.0–6.0, a stabilizing agent, and a non-ionic detergent aredisclosed in U.S. Pat. No. 5,151,265 issued 29 Sep. 1992. Thestabilizing agent typically is a polyhydric sugar alcohol, such asmannitol, and the non-ionic detergent may be a surfactant, e.g.polysorbate 80 or polysorbate 20. The non-ionic detergent preferably ispresent in a range of about 0.07 to 0.2 mg/ml, and most preferably in aconcentration of about 0.1 mg/ml. Suitable buffers are conventionalbuffers of organic acids and salt thereof, such as nitrate buffers(e.g., monosodium citrate-disodium citrate mixture, citricacid-trisodium citrate mixture, citric acid-monosodium citrate mixture,etc.), succinate buffers (e.g., succinic acid-monosodium succinatemixture, succinic acid-sodium hydroxide mixture, succinic acid-disodiumsuccinate mixture, etc.), tartarate buffers (e.g., tartaric acid-sodiumtartarate mixture, tartaric acid-potassium tartarate mixture, tartaricacid-sodium hydroxide mixture, etc.), fumarate buffers (e.g. fumaricacid-monosodium fumarate mixture, fumaric acid-disodium fumaratemixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconatebuffers (e.g. gluconic acid-sodium gluconate mixture, gluconicacid-sodium hydroxide mixture, gluconic acid-potassium gluconatemixture, etc.), oxalate buffers (e.g., oxalic acid-sodium oxalatemixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassiumoxalate mixture, etc.), lactate buffers (e.g. lactic acid-sodium lactatemixture, lactic acid-sodium hydroxide mixture, lactic acid-potassiumlactate mixture, etc.), and acetate buffers (e.g., acetic acid-sodiumacetate mixture, acetic acid-sodium hydroxide mixture, etc.)

A known commercial liquid formulation of IFN-γ (Actimmune® rhuIFN-γ-1b,Genentech, Inc.) is a sterile, clear, colorless unpreserved solutionfilled in a single-dose vial for subcutaneous injection. Each 0.5 mlvial of Actimmune contains 100 μg (3 million U, specific activity: 30million U/mg) of IFN-γ-1b formulated in 20 mg mannitol, 0.36 mg sodiumsuccinate, 0.05 mg polysorbate 20 and Sterile Water for Injection.

Preserved pharmaceutical compositions to be used in accordance with thepresent invention, which are suitable for repeated use, preferablycontain:

-   -   a) IFN-γ not subjected to prior lyophilization;    -   b) an acetate buffer capable of maintaining the pH between about        4 and about 6 (the pH range of maximum stability of the protein        in solution);    -   c) a non-ionic detergent primarily to stabilize the protein        against agitation-induced aggregation;    -   d) an isotonifier;    -   e) a preservative selected from the group of phenol, benzyl        alcohol and a benzethonium halide, e.g. chloride; and    -   f) water.

The non-ionic detergents (surfactants) may, for example, be polysorbates(e.g. polysorbate [Tween] 20, 80, etc.) or poloxamers (e.g. poloxamer188). The use of non-ionic surfactants permits the formulation to beexposed to shear surface stresses without causing denaturation of theprotein. Further, such surfactant containing formulations may beemployed in aerosol devices such as those used in a pulmonary dosing,and needleless jet injector guns (see, e.g. EP 257,956).

The isotonifier is present to ensure isotonicity of the liquidcompositions of the present invention, and includes polyhydric sugaralcohols, preferably trihydric or higher sugar alcohols, such asglycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Thesesugar alcohols can be used alone or in combination. Alternatively,sodium chloride or other appropriate inorganic salts may be used torender the solutions isotonic.

The acetate buffer may, for example, be an acetic acid-sodium acetatemixture, acetic acid-sodium hydroxide mixture, etc. The pH of the liquidformulation of this invention is buffered in the range of about 4.0 to6.0, preferably 4.5 to 5.5, and most preferably at about pH 5.

The preservatives phenol, benzyl alcohol and benzethonium halides, e.g.chloride, are known antimicrobial agents.

In a preferred embodiment, IFN-γ is administered in the form of a liquidpharmaceutical composition which comprises the following components:

IFN-γ 0.1–2.0 mg/ml sodium acetate (pH 5.0) 5–100 mM Tween 20 0.1 to0.01% by weight phenol 0.05 to 0.4% by weight mannitol 5% by weightwater for injection, USP up to 100%,wherein the percentage amounts are based on the weight of thecomposition. Phenol can be replaced by 0.5–1.0% by weight of benzylalcohol, and mannitol can be replaced by 0.9% by weight sodium chloride.

Most preferably, the compositions comprise

IFN-γ 0.1 to 1.0 mg/ml sodium acetate (pH 5.0) 10 mM Tween 20 0.01% byweight phenol 0.2% mannitol 5%

Phenol can be replaced by 0.75 by weight benzyl alcohol and mannitol by0.9% by weight sodium chloride.

The preserved liquid formulations preferably contain multiple doses of atherapeutically effective amount of IFN-γ. In view of the narrow hostrange of this polypeptide, for the treatment of human patients liquidformulations comprising human IFN-γ, more preferably native sequencehuman IFN-γ, are preferred. As a biological response modifier, IFN-γexerts a wide variety of activities on a wide range of cell types, in avariety of human and non-human mammalian species. The therapeuticallyeffective dose will, of course, vary depending on such factors as thepathological condition to be treated (including prevention), thepatient's age, weight, general medical condition, medical history, etc.,and its determination is well within the skill of a practicingphysician. The effective dose generally is within the range of fromabout 0.001 to about 1.0 mg/kg, more preferably about 0.01–1 mg/kg, mostpreferably about 0.01–0.1 mg/kg. In such formulations huIFN-γ willpreferably exhibit a specific activity of on the order of about 2×10⁷U/mg of protein or greater when tested on A549 cells againstencephalomyocarditis virus. It should be appreciated that endotoxincontamination should be kept minimally at a safe level, for example,less than 0.5 ng/mg protein. Moreover, for human administration, theliquid formulations should meet sterility, pyrogenicity, general safety,and purity as required by FDA Office and Biologics standards.

The route of IFN-γ administration is in accord with known methods, e.g.,injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial, or intralesional routes, orby sustained-release systems as noted below. Therapeutic IFN-γcompositions generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle. The formulationsare preferably administered as repeated intravenous (i.v.), subcutaneous(s.c.) or intramuscular (i.m.) injections, or as aerosol formulationssuitable for intranasal or intrapulmonary delivery (for intrapulmonarydelivery see, e.g. EP 257,956).

The stable aqueous compositions of IFN-γ are preferably contained invials, containing up to about 30 therapeutically effective doses ofIFN-γ. The bioactivity of IFN-γ preferably remains within about 20% fromthe bioactivity exhibited at the time of first administration for atleast about 14 days, more preferably for at least about 200 daysfollowing first administration.

IFN-γ can also be administered in the form of sustained-releasedpreparations. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe protein, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res., 15: 167–277 [1981]and Langer, Chem. Tech. 12: 98–105 [1982] or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22: 547–556 [1983]), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLupron Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated proteinsremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for protein stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

Sustained-release IFN-γ compositions also include liposomally entrappedIFN-γ. Liposomes containing IFN-γ are prepared by methods known per se:DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688–3692(1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030–4034 (1980);EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patentapplication 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP102,324. Ordinarily the liposomes are of the small (about 200–800Angstroms) unilamellar type in which the lipid content is greater thanabout 30 mol. % cholesterol, the selected proportion being adjusted forthe optimal therapy.

An effective amount of IFN-γ to be employed therapeutically will depend,for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the optimal therapeutic effect.The recommended dosage for the administration of IFN-γ (Actimmune®,Genentech, Inc.) to treat patients with chronic granulomatous disease is50 mcg/m² (1.5 million U/m²) for patients whose body surface area isgreater than 0.5 m², and 1.5 mcg/kg/dose for patients whose body surfacearea is equal to or less than 0.5 m², administered as a subcutaneousinjection, three times a week. This is valuable guidance for a physicianto determine the optimal effective dose for the treatment of cardiachypertrophy. The clinician will administer IFN-γ until a dosage isreached that achieves the desired effect for treatment of the heartdysfunction. For example, if the objective is the treatment ofcongestive heart failure, the amount would be one which inhibits theprogressive cardiac hypertrophy associated with this condition. Theprogress of this therapy is easily monitored by echo cardiography.Similarly, in patients with hypertrophic cardiomyopathy, IFN-γ can beadministered on an empirical basis, relying on the patient's subjectiveperception of benefit.

IFN-γ may be administered in combination with other therapeutic agentsused for the treatment (including prevention) of cardiac hypertrophy.For example, IFN-γ therapy can be combined with the administration ofinhibitors of known cardiac myocyte hypertrophy factors, e.g. inhibitorsof α-adrenergic agonists, e.g. phenylephrine; endothelin-1; CT-1; LIF;angiotensin converting enzyme; and angiotensin II. Inhibitors of cardiachypertrophy factor (CHF, cardiotrophin or cardiotrophin-1, see, e.g.U.S. Pat. No. 5,679,545) are particularly preferred for combinationtherapy.

Preferred candidates for combination therapy in the treatment ofhypertrophic cardiomyopathy are adrenergic-blocking drugs (e.g.,propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol,penbutolol, acetobutolol, atenolol, metoprolol, carvedilol), verapamil,difedipine, diltiazem. Treatment of hypertrophy associated with highblood pressure may require the use of antihypertensive drug therapy,using calcium channel blockers, e.g. diltiazem, nifedipine, verapamil,nicardipine; β-adrenergic blocking agents; diuretics, e.g.,chorothiazide, hydrochlorothiazide, hydroflumethazide,methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide,indapamide; and/or ACE-inhibitors, e.g., quinapril, captopril,enalapril, ramipril, benazepril, fosinopril, lisinopril.

The effective amount of the therapeutic agents administered incombination with IFN-γ will be at the physician's or veterinarian'sdiscretion. Dosage administration and adjustment is done to achieveoptimal management of the conditions to be treated, and ideally takesinto account use of diuretics or digitalis, and conditions such ashyper- or hypotension, renal impairment, etc. The dose will additionallydepend on such factors as the type of the therapeutic agent to be usedand the specific patient being treated. Typically, the amount employedwill be the same dose as that used, if the given therapeutic agent isadministered without IFN-γ.

EXAMPLES Example 1

Inhibition of PGF_(2α)-Induced Spreading Response of Adult Myocytes byIFN-γ

Materials and Methods

Adult myocyte cultures The procedure used for the isolation ofventricular myocytes from adult rats was a modification of a proceduredescribed by Piper et al., supra, and is detailed in Lai et al., supra.For each myocyte preparation, one male Sprague-Dawley rat weighing about250 g was anesthetized with pentobarbital sodium, and the heart wasremoved. Extraneous tissue was trimmed from the heart, and it wasmounted onto a Langendorff system that was temperature controlled at 37°C. The heart was perfused with about 40 ml of Krebs buffer (110 mM NaCl,2.6 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄.7H₂O, 25 mM NaHCO₃ and 11 mMglucose). A solution containing 30 mg of collagenase and 12.5 μl of 100mM CaCl₂ in 50 ml of Krebs buffer was then recirculated through theheart for 30 minutes. The heart was removed from the Langendorffapparatus, and the atria and connective tissues were removed. Theventricles were cut into 2 mm cubes with dissecting scissors, andfurther digested in a fresh collagenase solution (30 mg collagenase and400 mg BSA dissolved in Krebs buffer with 12.5 μl of 100 mM CaCl₂) forfive minutes at 37° C. During the digestion, the tissue suspension wasgently band shaken one time per minute. After the digestion, thesupernatant was removed and saved, and the remaining tissue was furtherdigested in fresh collagenase solution for an additional five minutes.

Isolated adult rat myocytes were plated on laminin-coated plates at adensity of 3×10³ cells/ml. After 72 hours of appropriate stimulation,the cells were fixed with gluteraldehyde and stained with Eosin Y.Images of rod shaped cells were captured under fluorescent microscopyand maximum breadth was determined using imaging software (Simple 32,Compix Imaging, Mars, Pa.).

Results

IFN-γ Inhibits the Spreading of Adult Cardiac Myocyte Induced by theHypertrophy Factors PGF_(2α) and Phenylephrine.

PGF_(2α), and the α-adrenergic agonist phenylephrine have been shown toinduce hypertrophy of cultured neonatal rat cardiac myocytes (Adams etal., J. Biol. Chem. 271: 1179–1186 [1996]; Lai et al., Am. J.Phvsiol.(Heart Circ. Physiol.) 271:H2197–H2208 [1996];Meidell et al.,Am. J. Physiol. 251:H1076–H1084 [1986]; Simpson, J. Clin. Invest.72:732–738 [1983]; Simpson, Circ. Res. 56:884–894 [1985]). Adult ratventricular myocytes spread when exposed to these factors in culture(Lai et al., supra; Piper et al., “Adult ventricular rat heart musclecells”, in: Cell Culture Techniques in Heart and Vessel Research, H. M.Piper, Editor, 1990, Springer-Verlag: Berlin, p. 36–60.) Adult myocyteshave a rod-like morphology. When these cells are exposed to 0.1 μMPGF_(2α), the rod shaped cells flatten and spread (FIG. 1). Thespreading response was quantified by measuring the maximum cell breadthof at least 200 rod shaped cells and plotting this value vs the percentfrequency of cell breadth in the population. PGF_(2α) induced asignificant change in the maximum cell breadth as evidenced by a shiftin the population distribution for this parameter compared to controlcells (P<0.001). Treating cells with IFN-γ significantly inhibited theirresponse to PGF_(2α) (P<0.001 PGF_(2α)+IFN-γ compared to PGF_(2α)). Theinhibitor effect of IFN-γ on PGF_(2α)-induced myocyte spreading was dosedependent (FIG. 2) over a concentration range that is consistent withthe biological response to IFN-γ in cardiac myocytes and other cellsystems (Singh et al, J. Biol. Chem. 271: 1111–1117 [1996]; Pinsky etal., J. Clin. Invest. 95:766–685 [1995]; Ungureanu-Longrois et al.,Circ. Res. 77:494–502 [1995]; Soderberg-Naucler et al., J. Clin. Invest.100:3154–3163 [1997]; Gou et al., J. Clin. Invest. 100:829–838 [1997];Marra et al., Can J. Cardiol. 12:1259–1267 [1996]). The ability toinhibit PGF_(2α), induced myocyte spreading appears to be specific toIFN-γ since several other cytokines including IL-1α, IL-1β, IL-2, IL-6,TNF-α, IFN-α, and IFN-β could not inhibit the spreading response. Theinhibitory effect of IFN-γ is not specific for PGF_(2α). IFN-γ can alsoinhibit spreading induced by phenylephrine (FIG. 3).

Example 2

Inhibition of Cardiac Hypertrophy In Vivo

Materials and Methods

Animals All experimental procedures conformed to the guiding principlesof the American Physiology Society, and were approved by Genentech'sInstitutional Animal Care and Use Committee. The animals used in thisstudy were male Sprague/Dawley (SD) rats (8 weeks of age, Charles RiverBreeding Laboratories, Inc.). The animals were acclimated to thefacility for at least one week before experiments, fed a pelleted ratchow and water ad libitum, and housed in a light and temperaturecontrolled room.

Administration of fluprostenol and/or IFN-γ Rats received subcutaneousinjection of fluprostenol (Cayman Chemical, Ann Arbor, Mich.) at 0.15mg/kg, recombinant mouse IFN-γ (Genentech, Inc., South San Francisco,Calif.) at 0.08 mg/kg, combination of fluprostenol and IFN-γ, or salinevehicle, twice a day for 14 days. In the IFN-γ and fluprostenol+IFN-γgroups, animals were pretreated with IFN-γ for one day. Body weight wasmeasured before and after treatment. A previous study has shown that thedose of fluprostenol used here is the lowest dose which produces asignificant cardiac hypertrophy in rats. Lai et al., supra. A pilotstudy demonstrated that IFN-γ at the dose indicated above inhibitedfluprostenol-induced cardiac hypertrophy with little effects on bodyweight in rats.

Hemodynamic assessment Thirteen days after treatment, rats wereanesthetized with intraperitoreal injection of ketamine 80 mg/kg (AvecoCo., Inc., Fort Dodge, Iowa) and xylazine 10 mg/kg (Rugby Laboratories,Inc., Rockville Center, N.Y.). A catheter (PE-10 fused with PE 50)filled with heparin-saline solution (50 U/ml) was implanted into theabdominal aorta, via the right femoral artery, for measurement of meanarterial pressure (MAP) and heart rate (HR). The catheter wasexteriorized and fixed at the back of the neck.

One day after catheterization, the arterial catheter was connected to aModel CP 10 pressure transducer (Century Technology Company, Inglewood,Calif., USA) that was coupled to a Grass Model 7 polygraph (GrassInstruments, Quincy, Mass., USA). MAP and HR were measuredsimultaneously in conscious, unrestrained rats.

Measurement of organ weights Under anesthesia with ketamine/xylazine,the heart, kidney, and spleen were removed, dissected and weighed. Theleft ventricle was stored at 80° C. for evaluation of gene expression.

Animal model of pressure overload The induction of pressure overload bypartial ligation of the abdominal aorta in rats was as describedpreviously. Kimura et al., Am. J. Physiol. 1989:256 (Heart Circ.Physiol. 25):H1006–H1-11; Batra et al., J. Cardiovasc. Pharmacol.17(suppl. 2), S151–S153 (1991). In brief, rats were anesthesized withketamine/xylazine as described above. A 3 cm midline incision was madein the abdominal wall. The abdominal aorta between the diaphragm and therenal artery was exposed and looped with 5–0 silk suture. The suture wastightened around a gauge 23 needle, and then the needle was withdrawn.Sham animals received the surgery without tightening the suture.

Experimental protocol in rats with pressure overload The rats withaortic banding randomly received subcutaneous injection of IFN-γ at 0.08mg/k twice a day for one day before surgery and for 14 days aftersurgery. Sham animals were not treated. Thirteen days after treatment, acatheter was implanted into the right carotid artery under anesthesia asindicated above. One day after implantation, arterial pressure and HRwere measured in conscious rats. The heart and other organs includingthe liver, kidney, and spleen were removed, weighed, and fixed in 10%buffered formalin for pathological studies. The left ventricle wasquickly dissected and frozen with liquid nitrogen in some animals andstored at −80° C. for assessment of gene expression.

Statistical analyisis Results are expressed as mean±SEM. One wayanalysis of variance (ANOVA) was performed to assess differences inparameters between groups. Significant differences were then subjectedto post hoc analysis using Newman-Keuls method; P<0.05 was consideredsignificant.

RNA preparation Total RNA was isolated using RNeasy Maxi Columns(Qïägen) according to the manufacturer's instructions.

RT-PCR Real-time RT-PCR (TaqMan) technology was used to compare the geneexpression between the various treatment groups. An oligonucleotideprobe containing a fluorescent reporter dye,6-carboxytetramethyl-rhodamine (TAMRA), at the 3′-end was designed tohybridize to the amplicon defined by two PCR primers. A 3′-blockingphosphate prevents extension of the probe. The reporter dye is releasedfrom the probe by the 5′ exonuclease activity of Taq polymerase duringthe extension phase of PCR reaction. The resulting fluorescence ismonitored in the reaction tube by the sequence detector and quantifiedwithout further manipulation, hence the term “real-time”. The thresholdcycle number (Ct), defined as the point where the reporter fluorescencereaches a value greater than 10 times the standard deviation of thebaseline, is proportional to the amount of amplicon produced from thesample. Since the fluorescence is detected during the exponential phaseof the amplification, none of the reaction components are limiting. Ineach experiment, a control is analyzed that lacks the RNA template tomonitor for contamination, and another control is included where the RTstep is omitted to eliminate amplification of possible contaminating DNAas the source of the signal. Reactions are optimized to give thegreatest fluorescence signal and smallest Ct by titration of magnesiumand primer concentrations, and the product is run on an agarose gel toverify the presence of a single band at the predicted molecular weight.In addition, the sequence of the amplicon is screened against Genbank toeliminate the possibility of overlap with closely related genes.

For each sample the mRNA for each target gene is determined using astandard curve as described below and then normalized to the amount ofglyceraldehyde-3-phosphate-dehydrogenase (GAPDH) in the sample (seebelow for specifics of this calculation). The relative abundance of eachtarget gene to GAPDH can then be compared among treatment groups.

RT-PCR was performed on 1 ng of total RNA per reaction using the TaqManModel 7700 Sequence Detector (ABI-Perkin Elmer) (Gibson et al., GenomeRes. 6, 995–1001 [1996]). Amplification reaction conditions (for 50 μl)were 1×TaqMan Buffer A, 200 μM dATP, dCTP, dGTP, and 400 μM dUTP, 10%glycerol, 6.5 mM MgCl₂, 50 U MuLV reverse transcriptase, 20 U RNaseInhibitor, 1.25 U AmpliTaq Gold, 100 nM forward and reverse primers, and100 nM fluorogenic probe. RT-PCR reagents and glycerol were purchasedfrom Perkin Elmer and Sigma, respectively. Reactions were performed inMicoAmp Optical Tubes and Caps (ABI-Perkin Elmer). TaqMan primers andprobes were designed according to guidelines determined by Perkin Elmerand synthesized at Genentech, Inc., except for those for rodent GAPDHwhich were a generous gift from Perkin Elmer. Reverse transcription wasperformed at 48° C. for 30 minutes followed by heat activation ofAmpliTaq Gold at 95° C. for 10 minutes. Thermal cycling was at 95° C.for 30 seconds and 60° for 1.5 minutes for 40 cycles.

Quantitation of the TaqMan results was performed as described by Heid etal., Genome Res. 6:986–994 (1996), with modifications. Briefly, standardcurves (1:5 serial dilution) for each target gene of interest were runin duplicate. The Ct was plotted on the Y axis vs the log of the totalRNA concentration (X axis), and the equation describing the line wasdetermined. MRNA for each target gene was determined from theappropriate standard curve by entering the Ct (Y value) and solving forthe input mRNA (X). The value for the target gene was then normalized toGAPDH by solving the following equation: 10^(X1)/10^(X2), where X1 isthe target gene, and X2 is GAPDH.

Results

IFN-γ Inhibits Cardiac Hypertrophy In Vivo

Chronic administration of fluprostenol, an agonist analog of PGF_(2α),has been shown to induce cardiac hypertrophy in vivo, and rats withpathologic cardiac hypertrophy induced by myocardial infarction havechronically elevated levels of extractable PGF_(2α) in their myocardium(Lai et al., supra). Thus, factors that can inhibit the effects ofPGF_(2α) on myocardial growth in vivo may be useful for treating cardiachypertrophy. Rats were dosed with fluprostenol in the presence andabsence of IFN-γ for two weeks, and the effects on cardiac hypertrophywere determined. Absolute weight of the heart, ventricles, and leftventricle tended to increase in the fluprostenol-treated rats, comparedto vehicle controls, and there was significant decrease in theseparameters in rats treated with fluprostenol+IFN-γ relative tofluprostenol treated rats (Table 1). Treatment with fluprostenolresulted in a significant increase in the ratio of heart, ventricular,and left ventricular weights to body weight (BW), indicating thatfluprostenol induced cardiac hypertrophy (FIG. 4). IFN-γ inhibitedfluprostenol induced hypertrophy. Rats receiving fluprostenol+IFN-γ hadsignificantly decreased heart, ventricular and left ventricular weight,normalized by BW, compared to animals in the fluprostenol groups (FIG.4). Comparison between the IFN-γ and vehicle treated groups showed thatadministration of IFN-γ alone did not significantly alter absolute orBW-normalized heat, ventricular, or left ventricular weights (Table 1,FIG. 4).

Chronic administration of fluprostenol was associated with a significantdecline in mean arterial pressure (MAP) compared to vehicle treatedcontrols (FIG. 5). IFN-γ had no effect on MAP compared to vehicle, anddid not affect the MAP of animals treated with fluprostenol. There wassignificant alteration in the heart rate in the four treatment groups(FIG. 5). These results indicate that IFN-γ did not inhibit hypertrophyinduced by fluprostenol by counteracting the hemodynamic effects of thetreatment.

IFN-γ not only inhibited the increase in cardiac mass associated withfluprostenol administration, but also the alterations in cardiac geneexpression associated with fluprostenol induced hypertrophy (FIG. 6).There was an increase in the abundance of mRNA for α-skeletal actin,collagen I, and natriuretic factor in the hearts of rats treated withfluprostenol compared to vehicle. The mRNA for sarcoplasmic reticulumcalcium ATPase was significantly reduced in these rats. IFN-γ inhibitedall but the atrial natriuretic factor response.

IFN-γ was also tested in a rodent model of cardiac hypertrophy inducedby pressure overload generated by abdominal aortic banding. Aorticconstriction resulted in cardiac hypertrophy as evidenced by substantialincreases in absolute heart, atrial, ventricular and left ventricularweights, and also the ratios of these weights to BW. Treatment withIFN-γ significantly attenuated cardiac hypertrophy in this model (Table2 and FIGS. 7 and 8).

The effect of IFN-γ on other organs was also examined (Table 2). Neitheraortic banding nor IFN-γ treatment altered kidney weight and the ratioof kidney weight to BW. Compared to sham-operated animals, liver weightand the ratio of BW tended to decrease in aortic-banded rats treatedwith vehicle, but not in those treated with IFN-γ. Aortic constrictioncaused a significant elevation in absolute and BW-normalized spleenweight, that was exaggerated by IFN-γ treatment. Thus, the effects ofIFN-γ on cardiac mass were not due to a generalized effect on organweight.

Mean arterial pressure, systolic pressure, and diastolic pressure weremarkedly higher in rats with aortic constriction compared tosham-operated controls, and the incremental increase in arterialpressure was not different between banded rats treated with IFN-γ orvehicle (FIG. 9). This result indicates that the attenuation of cardiachypertrophy observed in banded rats receiving IFN-γ did not relate to analteration in afterload.

Aortic constriction resulted in several changes in cardiac geneexpression. The relative abundance of mRNA for β-myosin heavy chain,α-smooth muscle and α-skeletal actins, atrial natriuretic factor,collagens I and III, and fibronectin were all increased in banded ratscompared to sham-operated controls. The effects on two of these genes;α-smooth muscle actin and collagen I were inhibited by IFN-γ (Table 3).

Taken together, the results in Examples 1 and 2 show that IFN-γ caninhibit cardia hypertrophy. The effects of IFN-γ are not limited toinhibiting an increase in cardiac mass induced by hypertrophic stimuli,IFN-γ can also inhibit certain of the molecular alterations that occurin the hypertrophied heart at the level of gene expression. It isespecially noteworthy that IFN-γ inhibited the induction of collagen Igene expression in vivo, both in response to chronic stimulation withfluprostenol and in a model of hypertrophy induced by pressure overload.Collagen I accounts for approximately 75% of myocardial collagen (Ju etal., Can. J. Caridol. 12:1259–1267 [1996]). Increased extracellularmatrix deposition and interstitial fibrosis that accompany cardiachypertrophy can contribute to the pathophysiology of heart failure. Byinhibiting collagen I production, IFN-γ may reduce interstitial fibrosisin the setting of heart failure.

TABLE 1 Body Weight and Organ Weight in Rats Treated with Flup and/orIFN Vehicle Flup Flup + IFN IFN BWO (g) 292.4 ± 1.7 292.3 ± 2.2 292.8 ±2.1 292.5 ± 3.2 BW (g) 391.6 ± 6.3 381.1 ± 4.4 377.6 ± 4.5 380.8 ± 6.1ΔBW (g)  99.2 ± 5.5  91.9 ± 4.0  84.8 ± 3.9  88.3 ± 5.0 HW (g)  .966 ±.022 1.000 ± .018 .9279 ± .016#  .956 ± .029 VW (g)  .922 ± .022  .957 ±.017  .889 ± .015#  .914 ± .029 LVW (g)  .706 ± .018  .740 ± .013  .678± .011##  .696 ± .023 KW (g) 1.440 ± .035 1.397 ± .038 1.377 ± .0311.327 ± .035* KW/BW 3.678 ± .075 3.632 ± .076 3.648 ± .070 3.483 ± .069(g/kg) SW (g)  .799 ± .050  .880 ± .048 1.009 ± .042*  .924 ± .068 SW/BW2.065 ± .149 2.309 ± .130 2.676 ± .082** 2.415 ± .149 (g/kg) Dataexpressed as mean ± SEM, and animal numbers are 14, 14, 14, and 9 in theVehicle, Flup, Flup + IFN, and IFN group, respectively. Vehicle, saline;Flup, fluprostenol; IFN, interferon γ, BWO, basal levels of body weight;BW, body weight post treatment; ΔBW, BW-BWO; HW, heart weight; VW,ventricular weight; LVW, left ventricular weight; KW, kidney weight; SW,spleen weight. *p <0.05, **P <0.01, compared to the Vehicle group, #p<0.05, ##p <0.01, compared to the Flup group.

TABLE 2 Body Weight, Organ Weight, and HR in Rats with Pressure OverloadSham PO + vehicle PO + IFN BWO (g) 278.8 ± 1.9 279.4 ± 1.3 279.0 ± 1.3BW (g) 367.9 ± 6.8 347.5 ± 5.7* 355.5 ± 5.2 ΔBW (g)  89.1 ± 5.8  68.1 ±5.6*  76.5 ± 4.7 AW (g)  .038 ± .002  .056 ± .002**  .046 ± .003*##AW/BW (g/kg)  .104 ± .004  .162 ± .006**  .129 ± .007*## KW (g) 1.438 ±.051 1.334 ± .033 1.349 ± .071 KWBW (g/kg) 3.894 ± .078 3.841 ± .0703.776 ± .071 LW (g) 13.84 ± .55 12.53 ± .36 13.96 ± .47# LW/BW (g/kg)37.46 ± .96 36.01 ± .71 38.94 ± .92# SW (g)  .724 ± .030  .839 ± .026*1.170 ± .053**## SW/BW (g/kg) 1.959 ± .051 2.418 ± .069** 3.261 ±.121**## HR (bpm)   371 ± 12   415 ± 12*   418 ± 19* Data expressed asmean ± SEM. Animal numbers are 16, 22, and 21 in the Sham, PO + vehicle,and PO + IFN group, respectively, for all parameters except HR for whichanimal number are 7, 8, and 7, respectively. PO, pressure overload; IFN,interferon γ; BWO, basal levels of body weight; BW, body weight posttreatment; ΔBW, BW-BWO; AW, atrial weight; KW, kidney weight; LW, liverweight; SW, spleen weight; HR, heart rate. *p <0.05, **P <0.01, comparedto the sham group. #p <0.05, ##p <0.01, compared to the PO + vehiclegroup.

TABLE 3 Effect of IFN on Gene Expression Treatment SHAM + Vehicle PO +Vehicle PO + IFN ANF 0.98 ± 0.37 5.29 ± 1.21† 3.61 ± 1.32 βMHC 0.89 ±0.24 1.91 ± 0.15† 1.71 ± 0.14† SKA 0.95 ± 0.13 3.35 ± 0.46† 2.47 ± 0.51†SMA 0.71 ± 0.06 0.89 ± 0.04† 0.77 ± 0.06 COLI 0.55 ± 0.05 0.91 ± 0.09†0.77 ± 0.10 COLIII 0.44 ± 0.05 0.66 ± 0.08† 0.72 ± 0.09† FIB 0.66 ± 0.171.03 ± 0.11† 0.97 ± 012 PO indicates pressure overload; IFN,interferon-gamma; ANF, atrial natriuretic factor; βMHC, β-myosin heavychain; SKA, α-skeletal actin; SMA, α-smooth muscle actin; COLI, collagenI; COLIII, collagen III; FIB, fibronectin. Expression levels arecalculated as ratios to glyceraldehyde-3-phosphate dehydrogenase. n = 6per group Values are mean ± SEM. †P <0.05 vs sham + vehicle group. †

1. A method of treating cardiac hypertrophy comprising administering toa patient diagnosed with pressure overload induced cardiac hypertrophy atherapeutically effective amount of interferon gamma (IFN-γ).
 2. Themethod of claim 1 wherein said patient is human.
 3. The method of claim2 wherein said IFN-γ is rhIFN-γ-1b.
 4. The method of claim 2 whereinsaid IFN-γ is administered in combination with at least one furthertherapeutic agent used for the treatment of cardiac hypertrophy or aheart disease resulting in cardiac hypertrophy.
 5. The method of claim 4wherein said further therapeutic agent is selected from the groupconsisting of β-adrenergic-blocking agents, verapamil, difedipine, anddiltiazem.
 6. The method of claim 5 wherein said β-adrenergic-blockingagent is carvedilol, propranolol, metaprolol, timolol, exprenolol ortertatolol.
 7. The method of claim 4 wherein said IFN-γ is administeredin combination with an antihypertensive drug.
 8. The method of claim 4wherein said IFN-γ is administered with an ACE-inhibitor.
 9. The methodof claim 4 wherein said IFN-γ is administered with an endothelinreceptor antagonist.
 10. The method of claim 4 wherein said IFN-γ isadministered following the administration of a thrombolytic agent. 11.The method of claim 10 wherein said thrombolytic agent is recombinanthuman tissue plasminogen activator (rht-PA).
 12. The method of claim 4wherein said IFN-γ is administered following primary angioplasty for thetreatment of acute myocardial infarction.